Gene targeting, homologous recombination between a newly added, exogenous DNA sequence and the cognate chromosomal sequence, in mouse embryo-derived stem (ES) cells allows the specific modification of any chosen genetic locus in living mice. First, a cloned copy of the chosen gene is mutated in vitro using standard recombinant DNA technology. The modified gene is then introduced into ES cells where homologous recombination transfers the mutation, created in a test tube, to the genome of the living cell. The ES cells carrying the mutant gene are then used to generate germline chimeras. Finally, intercrosses of heterozygous siblings are used to generate mice homozygous for the mutant gene. We propose to use this technology to genetically separate the functions of a complex multifunctional gene during mouse development. We hope to achieve this goal by identifying and modifying DNA cis elements responsible for mediating the complex expression pattern of the gene during development as well as by developing an inducible switch which will allow turning on or off the gene during different periods of development. In addition, we propose to use gene targeting to develop new approaches for cell lineage and mosaic analysis.